EP3954998B1 - Compositions and methods for detecting and treating gastric cancer - Google Patents

Compositions and methods for detecting and treating gastric cancer Download PDF

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EP3954998B1
EP3954998B1 EP21199550.1A EP21199550A EP3954998B1 EP 3954998 B1 EP3954998 B1 EP 3954998B1 EP 21199550 A EP21199550 A EP 21199550A EP 3954998 B1 EP3954998 B1 EP 3954998B1
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cdr
progastrin
preferentially
amino acid
antibody
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EP3954998A1 (en
EP3954998C0 (en
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Alexandre PRIEUR
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ECS Progastrin SA
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/595—Gastrins; Cholecystokinins [CCK]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the in vitro diagnosis, the prevention and the treatment of cancer, more particularly it relates to methods for the in vitro diagnosis of gastric cancer, and to methods and compositions for the prevention or the treatment of gastric cancer.
  • Compositions according to the invention comprise a progastrin-binding molecule, in particularly an anti-hPG antibody, whereas methods according to the invention comprise the use of a progastrin-binding molecule, and particularly to an anti-hPG antibody.
  • Gastric cancer or stomach cancer
  • gastric cancer is considered to be the fifth leading cause of cancer and the third leading cause of death from cancer making up 7% of cases and 9% of deaths.
  • the most common cause of gastric cancer is infection by the bacteria Helicobacter pylori, wherein studies have shown an association between Helicobacter pylori infection and gastric cancer.
  • Gastric cancer develops from the lining of the stomach, then the cancer may spread to other parts of the body, and in particular liver, lungs, bones, lining of the abdomen and lymph nodes.
  • Treatments usually include surgery, chemotherapy, radiation therapy, and targeted therapy, alone or in combination.
  • outcomes are often poor with a less than 10% 5-year survival rate globally. This is largely because most people are detected only with advanced disease, which has a direct consequence on the survival rate. In some Asian countries, screening efforts have shown to be associated with a higher survival rates.
  • Clinical diagnosis is based on biopsy, which is performed under endoscopy. Medical imaging can then be used to determine if the disease has spread to other parts of the body.
  • Henwood Henwood et al, 2001. Brit J Surg, 88(4), 564 - 568 ) discloses the expression of progastrin on in situ in gastric adenocarcinoma.
  • WO 2011/083091 relates to methods for treating pancreatic cancer.
  • the present invention provides a method for the in vitro diagnosis of gastric cancer in a subject, comprising the steps of:
  • step b) further comprises determining the concentration of progastrin and wherein a concentration of progastrin at least 10 pM, at least 20 pM, at least 30 pM or at least 40 pM in the biological sample is indicative of the presence of gastric cancer in the subject.
  • the gastric cancer is metastasized.
  • the method of the invention comprises the further steps of:
  • a gastric cancer is present if the concentration of progastrin in step b) is higher than the reference concentration of progastrin of step c).
  • the method further comprises a second diagnosis test comprising the detection of a particular biomarker chosen among: pepsinogen, ghrelin, trefoil factor 3 (TFF3) and circulating GC-associated antigen (MG7-Ag), preferably pepsinogen.
  • a biomarker chosen among: pepsinogen, ghrelin, trefoil factor 3 (TFF3) and circulating GC-associated antigen (MG7-Ag), preferably pepsinogen.
  • the present invention also relates to a method of monitoring the efficacy of a treatment for gastric cancer in a patient, comprising the steps of:
  • the progastrin-binding molecule is an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, wherein the antigen-binding fragment thereof comprises the 6 CDRs of the antibody from which it is derived.
  • the antibody, or antigen-binding fragment thereof is selected in the group consisting of:
  • the antibody binding to progastrin is a monoclonal antibody chosen in the group consisting of:
  • the binding of the progastrin-binding molecule to progastrin is detected and/or measured by Fluorescence Activated Cell Sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), western blot or immunohistochemistry (IHC), preferably by ELISA or RIA, more preferably by ELISA.
  • FACS Fluorescence Activated Cell Sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • IHC immunohistochemistry
  • the biological sample is contacted with a first molecule, which binds to a first part of progastrin, and with a second molecule, which binds to a second part of progastrin.
  • the present disclosure now provides methods for the in vitro diagnosis of gastric cancer, wherein said method comprises the detection progastrin in a biological sample from a subject. Preferably, the amount of progastrin in said sample is determined, thus allowing quantification of progastrin.
  • the present disclosure also provides a composition for use in the prevention or the treatment of gastric cancer, wherein said composition comprises an antibody binding to progastrin, and methods for the prevention or the treatment of gastric cancer comprising the use of a composition comprising an antibody binding to progastrin, alone or in combination with any other known prevention or therapeutic methods against gastric cancer.
  • Human pre-progastrin a 101 amino acids peptide (Amino acid sequence reference: AAB19304.1), is the primary translation product of the gastrin gene.
  • Progastrin is formed by cleavage of the first 21 amino acids (the signal peptide) from preprogastrin.
  • the 80 amino acid chain of progastrin is further processed by cleavage and modifying enzymes to several biologically active gastrin hormone forms: gastrin 34 (G34) and glycine-extended gastrin 34 (G34-Gly), comprising amino acids 38-71 of progastrin, gastrin 17 (G17) and glycine-extended gastrin 17 (G17-Gly), comprising amino acids 55 to 71 of progastrin.
  • Anti-human progastrin (anti-hPG) monoclonal antibodies and their use for diagnosis or therapy have been described in the following documents: WO 2011/083 088 for colorectal cancer, WO 2011/083 090 for breast cancer, WO 2011/083 091 for pancreatic cancer, WO 2011/116 954 for colorectal and gastrointestinal cancer, and WO 2012/013 609 and WO 2011/083089 for liver pathologies.
  • the present disclosure relates to a method for the in vitro evaluation of a risk of the presence of gastric cancer, wherein said method comprises a step of detecting progastrin in a biological sample from a subject.
  • the presence of progastrin in the sample indicates that there is a risk of the presence of gastric cancer.
  • the disclosure relates to an in vitro method for evaluating the risk of the presence of gastric cancer in a subject, said method comprising the steps of:
  • progastrin-binding molecule may be detected by various assays available to the skilled artisan. Although any suitable means for carrying out the assays are disclosed herein, it can be mentioned in particular FACS, ELISA, RIA, western-blot and IHC.
  • the method disclosed herein, but not part of the claimed invention, for the in vitro evaluation of a risk of the presence of gastric cancer in a subject comprises the steps of:
  • the concentration of progastrin present in the sample is determined, the result can be compared with those of control sample(s), which is (are) obtained in a manner similar to the test samples but from individual(s)s known not to suffer from a gastric cancer. If the concentration of progastrin is significantly more elevated in the test sample, it may be concluded that there is an increased likelihood that the subject from whom it was derived has a gastric cancer.
  • the method disclosed herein, but not part of the claimed invention comprises the further steps of:
  • the disclosure but not part of the claimed invention, relates to an in vitro method for diagnosing gastric cancer in a subject, said method comprising the steps of:
  • the present disclosure relates to a method for the in vitro diagnosis of gastric cancer in a subject, comprising the steps of:
  • a concentration of progastrin of at least 10 pM, at least 20 pM, at least 30 pM, in said biological sample is indicative of the presence of gastric cancer in said subject.
  • a preferred instance of the present disclosure relates to a method for the in vitro diagnosis of metastasized gastric cancer in a subject, from a biological sample of said subject, comprising the steps of:
  • a concentration of progastrin of at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM or at least 50 pM in said biological sample is indicative of the presence of metastasized gastric cancer in said subject.
  • the method disclosed herein, but not part of the claimed invention comprises the further steps of:
  • the present disclosure relates to a method for the in vitro diagnosis of gastric cancer in a subject, comprising the determination of the concentration of progastrin in a biological sample and comparing said value obtained to the concentration of progastrin in a reference sample.
  • evaluation of a risk of the presence of gastric cancer in a subject designates the determination of a relative probability for a given subject to suffer from gastric cancer, when compared to a reference subject or value.
  • a method according to the disclosure represents a tool in the evaluation of said risk, in combination with other methods or indicators such as clinical examination, biopsy and determination of the level of a known biomarker of gastric cancer, such as, for example, pepsinogen.
  • in vitro diagnosis means to determine if a subject is suffering from a particular affection. It is known that the diagnosis of gastric cancer involves at least a clinical observation of the symptoms of said subject and of the detection of pepsinogen. Pepsinogen testing is currently used as a biomarker but the accuracy of this test to detect gastric cancer is low, with sensitivity estimates ranging from 35.8% to 62.3% ( Yanaoka et al, Cancer Epidemiol Biomarkers Prev, 2008 ). Although some biomarkers were identified in the discovery phase, it is still a major challenge to transfer them into the clinic, mostly because of the lack of a systematic evaluation process ( Pepe et al, J Natl Cancer Inst, 2008 ).
  • a method for the in vitro diagnosis of gastric cancer can be considered as a tool within a diagnosis process.
  • the present disclosure relates to a method for the in vitro diagnosis of gastric cancer in a subject, comprises the determination of the concentration of progastrin in said biological sample and the determination of a known biomarker of gastric cancer, preferably pepsinogen.
  • gastric cancer also designates “stomach cancer”, it includes in particular “gastric carcinomas”, but also lymphomas and mesenchymal tumors which may also develop within the stomach.
  • gastric cancer also involves gastric cancer associated with metastasis, in particular liver, lungs, bones, abdomen and lymph nodes metastasis.
  • progastrin designates the mammalian progastrin peptide, and particularly human progastrin.
  • human progastrin refers to the human PG of sequence SEQ ID No. 1.
  • Human progastrin comprises notably a N-terminus and a C-terminus domains which are not present in the biologically active gastrin hormone forms mentioned above.
  • sequence of said N-terminus domain is represented by SEQ ID NO. 2.
  • sequence of said C-terminus domain is represented by SEQ ID NO. 3.
  • obtaining a biological sample it is herein meant to obtain a biological sample for use in methods described herein. Most often, but not part of the claimed invention, this will be done by removing a sample of cells from an animal but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the present methods in vivo., albeit not part of the claimed invention. Archival tissues, having treatment or outcome history, will be particularly useful.
  • the in vitro diagnosis method of a gastric cancer disclosed herein comprises the determination of the concentration of progastrin in a biological sample from a subject, wherein said subject exhibits at least one clinical symptom of gastric cancer.
  • the biological sample is chosen among: blood, serum and plasma.
  • the present disclosure relates to an in vitro diagnosis method of a gastric cancer comprising the determination of the concentration of progastrin in a biological sample from a subject, wherein said subject exhibits at least one clinical symptom of cancer and/or of metastasis.
  • binding By “binding”, “binds”, or the like, it is intended that the antibody, or antigen binding fragment thereof, forms a complex with an antigen which, under physiologic conditions, is relatively stable.
  • Methods for determining whether two molecules bind include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • said antibody, or antigen-binding fragment thereof binds to progastrin with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule such as BSA or casein. More preferably, said antibody, or antigen-binding fragment thereof, binds only to progastrin.
  • a biological sample from the subject is contacted with at least one progastrin-binding molecule, wherein the affinity of said molecule for progastrin is of at least 100 nM, at least 90 nM, at least 80 nM, at least 70 nM, at least 60 nM, at least 50 nM, at least 40 nM, at least 30 nM, at least 20 nM, at least 10 nM, at least 5 nM, at least 1 nM, at least 100 pM, at least 10 pM, or at least 1 pM, as determined by a method such as above-described.
  • the method for the diagnosis of gastric cancer described herein comprises the detection of the concentration of progastrin in a biological sample from a subject, wherein said biological sample is contacted with an anti-hPG antibody, or an antigen-binding fragment thereof.
  • the biological sample is chosen among: blood, serum and plasma.
  • antibody as used herein is intended to include polyclonal and monoclonal antibodies.
  • An antibody (or “immunoglobulin”) consists of a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR) or “hypervariable regions”, which are primarily responsible for binding an epitope of an antigen, and which are interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • CDRs means the hypervariable regions of the heavy and light chains of an antibody as defined by IMGT, wherein the IMGT unique numbering provides a standardized delimitation of the framework regions and of the complementary determining regions, CDR1-IMGT: 27 to 38, CDR2.
  • a method disclosed herein comprises contacting a biological sample with an anti-hPG antibody binding to an epitope of hPG, wherein said epitope includes an amino acid sequence corresponding to an amino acid sequence of the N-terminal part of progastrin chosen among an amino acid sequence corresponding to amino acids 10 to 14 of hPG, amino acids 9 to 14 of hPG, amino acids 4 to 10 of hPG, amino acids 2 to 10 of hPG and amino acids 2 to 14 of hPG, wherein the amino acid sequence of hPG is SEQ ID N° 1.
  • a method disclosed herein comprises contacting a biological sample with an anti-hPG antibody binding to an epitope of hPG, wherein said epitope includes an amino acid sequence corresponding to an amino acid sequence of the C-terminal part of progastrin, chosen among an amino acid sequence corresponding to amino acids 71 to 74 of hPG, amino acids 69 to 73 of hPG, amino acids 71 to 80 of hPG (SEQ ID N°40), amino acids 76 to 80 of hPG, and amino acids 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQ ID N°1.
  • the present method for the diagnosis of gastric cancer comprises the detection of progastrin in a biological sample from a human subject.
  • the method of the present disclosure comprises contacting a sample from said subject with an anti-hPG antibody as described above, wherein a concentration of progastrin superior to 10 pM, 20 pM, 30 pM or 40 pM in said sample is indicative of the presence of gastric cancer in said subject.
  • the method disclosed herein comprises contacting a sample from said subject with an anti-hPG antibody as described above, wherein a concentration of progastrin superior to 10 pM, preferably to 20 pM, more preferably to 30 pM, still more preferably to 40 pM, even more preferably to 50 pM in said sample is indicative of the presence of metastasized gastric cancer in said subject
  • the present disclosure also relates to methods for monitoring the efficacy of a treatment for gastric cancer in a patient, such as chemotherapy, biological therapy, immunotherapy or antibody therapy, by determining the concentration of progastrin in a first sample, such as a bodily fluid or biopsy of gastric cancer, obtained from a patient before treatment for gastric cancer, and then comparing the concentration of progastrin in the first sample to that in a second sample obtained from the same patient after treatment, where a reduction in the concentration of progastrin in said second sample compared to said first sample indicates that the treatment was effective.
  • the biological sample is chosen among: blood, serum and plasma.
  • a method disclosed herein comprises the determination of the level of progastrin over time in samples from a patient who has been or is being treated for gastric cancer.
  • Example 1 Detection of plasmatic progastrin concentration using polyclonal antibodies
  • Plasma progastrin levels were quantified by ELISA through the use of two specific anti-progastrin antibodies: capture antibodies are coated on the wells of the plate, whereas revelation antibodies are used to detect progastrin and mediates revelation of the signal.
  • quantification is based on the ELISA method which allows, through the use of a substrate whose reaction emits light, to assign a value proportional to the luminescence amount of antibodies bound to the antigen retained by capture antibodies.
  • binding characteristics of polyclonal antibodies against progastrin used in this assay are the following: absence of binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.
  • 96 wells plates are coated by preparing a solution of carbonate - sodium bicarbonate, 50 mM pH 9.6 by dissolving the contents of one capsule in 100 ml of MilliQ water.
  • a solution of capture antibody (3 ⁇ g / ml ), corresponding to polyclonal antibodies obtained by using the C-terminal of progastrin FGRRSAEDEN (SEQ ID N°40) is prepared in carbonate buffer. 100 microliters of antibodies solution is added to each well and incubated at 4 ° C for 16 hours (1 night).
  • Plates are then blocked by eliminating the antibodies solution and wash 3 times with 300 ⁇ l 1X PBS / 0.1% Tween-20, then adding 200 ⁇ l of blocking buffer (1X PBS / 0.1% Tween-20 / 0.1% BSA) per well, and incubated 2 hours at 22°C. Blocking buffer is then eliminated, wells are washed 3 times with 300 ⁇ l 1X PBS / 0.1% Tween-20.
  • Plasma dilution is performed as follows: The plasma is used pure, diluted 1/2, 1/5 and 1/10. Dilutions are prepared from pure plasma in 1X PBS / 0.1% Tween 20 / 0.1% BSA.
  • progastrin dilution is prepared as follows: stock recombinant PG (Full length human progastrin produced in E. coli and affinity purified with Glutathione agarose/Tag removal (Tev)/IMAC Counter purification/dialysis, from Institut Pasteur, Paris, France) is prepared at a concentration of 0.45 mg/ml (45 microM), in triplicate. Ranges of progastrin concentrations were prepared as follows:
  • the range of recombinant PG is linear and can therefore be more or less extensive according to the antibody used.
  • test samples approximately 500 ⁇ l of each sample are set aside and stored until analysis (and confirmation if necessary) of the results. 100 ⁇ l of each point of the range and/or plasmas are assayed pure, diluted to 1/2, 1/5 and 1/10, and incubated for 2 hours at 22 ° C on the plates.
  • the plates are washed 3 times with 300 ⁇ l 1X PBS / 0.1% Tween-20.
  • streptavidin-HRP The revelation with streptavidin-HRP is performed by removing detection antibody and wash 3 times with 300 ⁇ l 1X PBS / 0.1% Tween-20, then preparing a solution of Streptavidin-HRP at 20 ng / ml diluted in 1X PBS / 0.1% Tween-20 / 0.1% BSA, wherein 100 Add 100 ⁇ l of this solution is added to each well, before incubation for 1 hour at 22 ° C.
  • progastrin-specific antibody The wells of Nunc MaxiSORP 96-well plates are coated with a first progastrin- specific antibody as follows. Anti-progastrin monoclonal antibodies specific for the carboxy- terminal region of progastrin are diluted to a concentration of 3 ⁇ g/ml in a solution of 50 mM, pH 9.6 sodium carbonate/bicarbonate buffer in MilliQ water.
  • the second standard curve which serves as a negative control, is prepared from progastrin-negative human plasma diluted in blocking buffer at the same dilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10.
  • Said mAb is a C-terminal anti-hPG antibody, selected among:
  • the number of cells at T0 is counted in a control well, for each experiment.
  • the number of live cells in both control and anti-hPG mAb treated wells is counted at 48 hours, then the difference between each cell count and the cell count determined at T0, is calculated. The resulting number of anti-hPG mAb-treated cells is then expressed as a percentage of the number of control mAb-treated cells.
  • Humanized antibodies to PG are tested for their ability to inhibit proliferation of KATO-III, AGS, MGC-803, and SNU-1 cell lines. Survival of cells from each KATO-III, AGS, MGC-803, and SNU-1 cell line is tested using different anti-hPG humanized antibodies.
  • 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting at 24 hours after seeding (time "T0"), cells are treated in sextuplicates every 12h for 48 hours, in the absence of fetal calf serum, with 1 to 20 ⁇ g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 ⁇ g/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody. The number of cells at T0 is counted in a control well, for each experiment.
  • Treatment with anti-hPG Hz antibodies reduces cell number as compared to treatment with control antibody.
  • anti-hPG antibodies reduce cell survival.
  • Monoclonal antibodies to PG are tested for their ability to reduce cancer stem cell (CSC) frequency in KATO-III, AGS, MGC-803, and SNU-1 cell lines using Extreme Limiting Dilution Assay (ELDA).
  • CSC frequency from each KATO-III, AGS, MGC-803, and SNU-1 cell line is tested using different anti-hPG monoclonal antibodies.
  • the plates are observed with a phase-contrast microscope and the number of positive wells per cellular concentration is assessed.
  • the ELDA webtool http://www.bioinf.wehi.edu.au/software/elda/) is used to calculate the CSC frequencies of each treatment group and test for any statistical difference between groups (modified Chi-square test).
  • Treatment with anti-hPG monoclonal antibodies reduces CSC frequency as compared to treatment with control antibody.
  • Humanized antibodies to PG are tested for their ability to reduce cancer stem cell (CSC) frequency in in KATO-III, AGS and SNU-1 cell line using sphere formation assay.
  • CSC cancer stem cell
  • the wells are photographed via brightfield microscopy, the pictures are analyzed and the spheres with a mean diameter above 25 ⁇ m are counted.
  • Treatment with anti-hPG humanized antibodies reduces CSC frequency as compared to treatment with control antibody.
  • CSC cancer stem cell
  • cells are seeded in ultra-low attachment (ULA) P96 (96-well plates) at fixed cellular concentrations per well using a FACS Aria flow cytometer, and a range of concentrations is used from one to 500 cells per well.
  • the cells are cultivated for up to 11 days in ULA plates with M11 medium ( Macari et al, Oncogene, 2015 ) and treated every 3 or 4 days with 1 to 20 ⁇ g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 ⁇ g/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.
  • ULA ultra-low attachment
  • the plates are observed with a phase-contrast microscope and the number of positive wells per cellular concentration is assessed.
  • the ELDA webtool http://www.bioinf.wehi.edu.au/software/elda/) is used to calculate the CSC frequencies of each treatment group and test for any statistical difference between groups (modified Chi-square test).
  • Treatment with anti-hPG humanized antibodies reduces CSC frequency as compared to treatment with control antibody.
  • KATO-III, AGS, MGC-803, and SNU-1 are cell lines commonly used to study gastric cancer, which produce and secrete progastrin. Monoclonal antibodies to PG were tested for their ability to inhibit the WNT/ ⁇ -catenin pathway in these different cell lines using the expression of the protein survivin, a well-known WNT/ ⁇ -catenin pathway targeted gene, as read-out. Survivin expression from each KATO-III, AGS, MGC-803, and SNU-1 cell line is tested using different anti-hPG monoclonal antibodies.
  • 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting 24 hours after seeding cells are treated in quadruplicate every 12h for 72 hours, in the absence of fetal calf serum, with 1 to 20 ⁇ g/ml of monoclonal control antibodies (monoclonal antiboby anti-puromycin)(CT mAb), or with 1 to 20 ⁇ g/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.
  • CT mAb monoclonal antiboby anti-puromycin
  • Treatment with anti-hPG monoclonal antibodies reduces survivin expression as compared to treatment with control antibody.
  • Humanized antibodies to PG are tested for their ability to inhibit the WNT/ ⁇ -catenin pathway in KATO-III, AGS, MGC-803, and SNU-1 cell lines using the expression of the protein survivin, a well-known WNT/ ⁇ -catenin pathway targeted gene, as read-out.
  • Survivin expression from each KATO-III, AGS, MGC-803, and SNU-1 cell line is tested using different anti-hPG humanized antibodies.
  • 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting 24 hours after seeding cells are treated in quadruplicate every 12h for 72 hours, in the absence of fetal calf serum, with 1 to 20 ⁇ g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 ⁇ g/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.
  • humanized control antibodies anti-human FcG1, from BioXCell
  • Treatment with anti-hPG humanized antibodies reduces survivin expression as compared to treatment with control antibody.

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